The overall goal of this grant is to develop improved methods of treating axial back pain via intervertebral disc tissue engineering. Our previous research indicates that disc degeneration is a manifestation of poor nucleus cell function. Adult mesenchymal stem cells (MSCs) are an attractive cell source for nucleus rejuvenation, however, their differentiation and function need to be optimized to thrive in the challenging degenerate disc environment. To improve MSC resilience in vivo, we discovered a novel co-culture system where MSCs are packaged in a spherical configuration with chondrocytes. This bi-laminar structure allows for homotypic and heterotypic cellular interactions that mimic the developmental processes of condensation (where cell aggregates form) and induction (where a mature tissue layer directs the differentiation of a nave one). Our ongoing studies demonstrate that bi-laminar cell pellets (BCPs) have superior matrix synthesis and gene expression under in vitro conditions like those of the degenerate disc. Given the promising BCP results, we now propose in Aim 1 to optimize a small BCP configuration to for minimally-invasive intradiscal injection. In Aim 3 we will determine whether optimized BCPs lead to morphologically-relevant improvements (as compared to appropriate control conditions) within a small animal model of disc injury. PUBLIC HEALTH RELEVANCE: The goals of this new R21 application are to: 1) optimize a stem cell therapy for intervertebral disc degeneration; and 2) demonstrate disease modifying activity in a small animal model. This research will have substantial impact by providing a minimally- invasive therapy for the management of patients with the most common and costly musculoskeletal condition - chronic low back pain. The proposed therapeutic approach may also have future applications for treatment of cartilage and bone conditions, such as osteoarthritis and osteonecrosis.